Tetracycline process



United States Patent TETRACYCLINE PROCESS Fred W. Tanner, Jr., Baldwin, N.Y., assignor to Chas. Pfizer & Co. Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed Mar. 22, 1957, Ser. No. 647,741

6 Claims. (Cl. 195-80) This application is concerned with a new and useful process for the production of tetracycline. More par ticularly, it is concerned with a process by which tetracycline is preferentially produced by the addition of a chemical agent to a fermentation medium which in the absence of this agent produces a major proportion of or event only chlortetracycline.

Tetracycline is a broad'spectrum antibiotic the therapeutic efiicacy of which has been attached by a large number of medical investigators as reported in many articles in the medical literature. Its chemical structure was first reported in the Journal of the American Chemical Society, volume 74, page 4976 (1952). Its chemical relationship to chlortetracycline and oxytetracycline is well known.

Tetracycline is generally produced by one of two methods. These methods are catalytic dehalogenation of chlortetracycline with hydrogen, and fermentation. In both of these methods, there are definite disadvantages. The catalytic dehalogenation of chlortetracycline requires that the antibiotic be isolated from a fermentation source in relatively high purity and then subjected to chemical action requiring expensive processing equipment and catalysts. The fermentation method, although economically more practical, produces not only tetracycline but also chlortetracycline. In most media the latter is generally produced preferentially. This necessitates expensive purification procedures to separate the two antibiotics.

Because of its high activity and because of its high stability in certain media, tetracycline is preferred in many applications over other antibiotics. It is apparent,

therefore, that any process which makes it possible to produce tetracycline substantially uncontaminated by chlortetracyline or without the necessity of chemical processing fulfills a definite and long-felt need in the art.

The usual tetracycline-producing microorganisms belong to the genus Streptomyces. The particular strains which are usually used are Streptomyces aureofaciens, for example, Streptomyces aureofaciens (NRRL 2209), and Streptomyces viridifaciens, for example,Streptomyces viridifaciens (ATCC 11989). These microorganisms produce fermentation products containing substantially only chlortetracycline. Many attempts have been made to alter the usual fermentation media in order to suppress chlortetracycline in favor of tetracycline in the produced antibiotic mixture. Two of these are described in the patent literature. U.S. Patent 2,739,924 issued to Lein and Gourevitch describes the addition of a bromide and the like, for example, sodium bromide to fermentation media. U.S. Patent 2,734,018 issued to Minieri et al. describes the use of a dechlorinated fermentation media. While both of the described processes are capable of producing broths of increased tetracycline content, neither of them is completely satisfactory in that the amount of chlortetracycline produced is still relatively high. Furthermore, the dechlorination procedure is laborious and expensive.

It has now most unexpectedly been discovered that the addition of Z-chIoro-benzoxazole or Z-chloro-benzothiazole surprisingly makes it possible to produce tetracycline and chlortetracycline in mixtures containing as high as 98% tetracycline or even higher. The 2-chlor0- benzoxazole 'or 2-chloro-benzothiazole may be substituted on the carbon atoms of the phenyl ring with halogen atoms or with such groups as lower alkyl, hydroxyl, nitro, mercapto and others, but the unsubstituted compound is highly active per se. Other variations in the structure of the basic molecule may be made without seriously interfering with the desirable activity.

In practicing the instant invention a 2-chloro-benzoxazole or 2-chloro-benzothiazole is added to a fermentation broth of a tetracycline-producing microorganism of the genus Streptomyces and aerobic fermentation is allowed to continue in the normal way. The fermentation may be carried out in accordance With either the well known submerged or surface techniques. When the antibiotic activity of the fermentation broth is isolated, for example, by extraction and the antibiotic content analyzed, it is found that the amount of tetracycline produced is substantially higher than is produced in the absence of either of these agents. So surprisingly efficient is the process of this invention that concentrations of the tetracycline-producing agent as low as twently parts per million produce broths containing up to 200 g/ml. of tetracycline of greater than purity. The activity and purity of the tetracycline produced were established by chromatographic methods in which known solutions of tetracycline and chlortetracycline and mixtures of these were used as referen standards. Control experiments run under exactly the same conditions and evaluated in exactly the same manner produced broths containing 200 ng/ml of chlortetracycline and only a trace of tetracycline. Similarly high ratios of tetracycline to chlortetracycline are achieved with concentrations of 2-chloro-benzoxazole or 2-chloro-benzothiazole as low as 0.2 part per million or even lower. With most chlortetracycline producing microorganisms concentrations of about twenty parts per million are entirely adequate but the optimum concentration may vary somewhat with the particular strain or mutant, of microorganism being used. Thus certain microorganisms may yield optimum amounts of tetracycline at concentrations higher or lower than twenty parts per million. It is conceivable that with some particular strains or mutants the toxicity level may be higher or lower than with the microorganisms heretofore tested. The optimum concentration may depend in some cases on whether the 2- chloro-benzoxazole or 2-chloro-benzothiazole is added before or after inoculation of the fermentation media. For most applications, however, concentrations as high as 100 parts per million or even higher are tolerable. It is to be noted that the desirable effect of obtaining extremely high tetracycline to chlortetracycline ratio in the produced antibiotic is achieved without the necessity of expensive dechlorination procedures being applied to the fermentation media. 7 It is a most unusual finding to discover that either 2- chloro-benzoxazole or Z-chloro-benzothiazole aids in the production of tetracycline, even more so when it is considered that 2-dehalogenated benzoxazole' and benzothiazole do not produce the desirable effect. It is certainly most unexpected to find that the presence of chlorine on the-chemical agent is required to produce a nonhalogenated antibiotic, whereas a non-halogenated chemi- -cal agent produces a halogenated antibiotic.

' derantly chlortetracycline.

' Corn starch 1 Magnesium sulfate heptahydrate Calcium carbonate pounds per steam pressure.

a in a 300 ml. Erlenmeyer media, will usually contain a source of carbohydrates and a, source of nitrogen preferably in organic form. A; variety of diiierent metallic salts also have some value in stimulating production of the antibiotic. Among the carbohydrate; solutions which'are useful are molasses, lucosestarches glycerol, etc. I Qrga-nic nitrogen'sources include soybean meal, Wheat gluten, peanut meal, hydrol yzate ofcasein' and other proteins. Certain crude materials contain growth stimulatoryi'substanceswhich areof somevalue in obtaining maximum yields ofthe anti-' biotic. "These include such substances as corn steep liquoni distillers solubles,yeast extract, etc. Salts such assodiumnitrate, sodium chloride, potassium phosphate and magnesium sulfate can also be used. Occasionally the addition of a buffering agent such as calcium car, bonate is.helpful and if foaming is encountered during the growth of the organisms the addition of oils such as lard oil, soybean oil, etc. may be advisable. media. are described in US. Patents 2,482,055, 2,709,672 and 2,776,243 as well as in US patent applications, Serial tary shaker, approximately 2.5 m1. of this culture served Useful Nos. 391,708, 391,709 "and 391,710, filed November 12, v

1953, now abandoned.

In practicing the instant invention the active agent may be added to the fermentation media before or after inoculation. It is, however, preferably added before significant amounts of antibiotic activity have been produced since prior to the'a'ddition of 2-chloro-benzoxaz'ole or 2- chloro-benzothiazole the antibiotic produced is prepoib" The following examples are given solely for the purpos'e'of illustrationonly and are not. to be construed as limitations ofthis invention, many apparent variations of which are possible'without' departing from the spirit or scope thereof. V, A f Example I A spore suspension of Streptomyces aureofaciens was introduced into 100 ml. of the following composition in ,a 300 ml. Erlenmeyerfiask.

l i a Grams per liter Sucrose I 10 Corn steep liquor Ammonium monohydrogen sulfate 2 Calcium carbonate 2 -After forty-eight hours incubation at 28 on a rotary the Z-chlorobenzothiazole after incubation and the incubation was continued forau shaker, approximately 2.5 ml. of this culture served to,

inoculate one liter of a fermentation medium of the following composition. I I V 7 g. per liter. g. per liter. 5 g. per liter.

Corn steep liquor u Ammonium sulfate 7 7 'g. per liter. Z-chloro-benzoxazole 20 mg./l

tographic methods for antibiotic content. It was found that the broth contained 200 i /m1. .oftetracycline and that-this represented more than 98% of the total anti: biotic activity produced. The remainder ofv the antibiotic activity was attributed to chlortetracycline.

Example I] A sporejsuspension of Streptomyces aureofaciens was introduced into 100 m1. of the following composition Grams per liter After fo tY-e ght hours incubation at 28C. on a ro-' arcc 11989) was inoculated as 2 g. perliter.

0f the broth were withdrawn.- and tested by "paper chroma- I 1 200 tug/ml. of about (NRRL used to inoculate 100 having the following composition.

to inoculate one liter of a fermentation medium of the following composition. l

Corn starch Q. 20 g. per liter. Corn steep liquor 25 g. per liter. Ammonium sulfate 5 g. per liter. Magnesium sulfate heptahydrate. 2 g. per liter. Calcium carbonate .I i 7 g. per liter. 2-chloro-benztlthiazolcgn; f .i 2'0 mg./l.

Example 111 The procedure of Example I was repeated except that the 2-chlorobenzoxazole was added twenty-four l 1 ours after incubation and the incubation was continued for an additional sixty-six hours at 28 C. Analysis 'ofaliquot portions of. the broth established a tetracycline content of purity.

Example IV The procedure of Example'II was repeated except that was added twenty-four hours additional sixty-six hours at 28 C. Analysis of aliquot portions of the broth established a tetracycline content of 200 lg/ml. of. about 95 purity. V

I I Example V.

A spore suspension of Streptomyces viridifaciens (ATCC 11989) was inoculated as described in Example I. The medium used was identical with that described in Example I, and it was incubated for hours at 28 C. on a rotary shaker.

Analysis of aliquot portions of the broth established a tetracycline content of 200 g/ ml. of about 97% purity. 7

Example VI i A' spore suspension .of Streptomyces viridifacz'cr s described in Example II. The medium used was identical with that described Was incubatedfor 100 hours at 28 Analysis of aliquot portions of the in Example II, and it C. on a rotary shaker.

broth established. a tetracycline content of 200 ug/ml. of

about 97% purity. V V V V 7 Example VII A spore suspension of Streptamyces cureofacienr 2209) prepared as describedjin Example'l' was ml. of a sterile nutrient medium Sodium nitrate 3 2-chloro-benzoxazole m "he 85 100 ml. of tap water.

contained 200 ug/ml. of tetracycline and that this represented more than 98% of the total antibiotic activity producedJ 1 Example VIII A spore suspension of Streptomyces aw'eafacr'erts '(NRRL 2209) prepared as described in Example! was Crrarnsperliter 'Soybean meal V ;4

used to inoculate 100 ml. of a sterile nutrient medium having the following composition.

Grams per liter Soybean meal 40 Sodium nitrate 3 2-chloro-benzothiazole 85 100 ml. of tap water.

The medium was 'autoclaved forty minutes at twenty pounds steam pressure. After 100 hours incubation at 35 C. in a rotary shaker, samples were withdrawn and tested for antibiotic activity. It was found that the broth contained 200 rig/ml. of tetracycline and that this represented more than 98% of the total antibiotic activity produced.

Example IX A spore suspension of Streptomyces viridifaciens prepared as in Example I was used to inoculate 100 ml. of a sterile nutrient medium having the following composition.

Soybean meal 40 g. per liter. Sodium nitrate 3 g. per liter. 2-chloro-benzoxazole 2 mg. per liter 100 ml. of tap water.

The medium was autoclaved forty minutes at twenty pounds steam pressure. After 100 hours incubation at 35 C. in a rotary shaker, samples were withdrawn and tested for antibiotic activity. It was found that the broth contained 200 pg/ml. of tetracycline and that this represented more than 90% of the total antibiotic activity produced.

ExampleX A spore suspension of Streptomyces viridifaciens prepared as in Example I was used to inoculate 100 ml. of a sterile nutrient medium having the following composition.

Soybean meal 40 g. per liter. Sodium nitrate 3 g. per liter. 2-chloro-benzothiazole 2 mg. per liter 100 ml. of tap water.

ganism of the genus Streptomyces, the improvement which comprises conducting the fermentation in the presence of a compound selected from the group consisting of 2- chloro-benzoxazole and 2-chloro-benzothiozole, whereby 5 the production of chlortetracycline is suppressed in favor of tetracycline.

2. In a process for producing tetracycline by aerobic fermentation with a chlortetracycline-producing microorganis of the species Streptomyces aureofaciens, the improvement which comprises conducting the fermentation in the presence of a compound selected from the group consisting of 2-chloro-benzoxazole and 2-chloro-benzothiozole, whereby the production of chlortetracycline is suppressed in favor of tetracycline.

3. In a process for producing tetracycline by aerobic fermentation with a chlortetracycline-producing microorganism of the species Streptomyces viridifaciens, the improvement which comprises conducting the fermentation in the presence of a compound selected from the group consisting of 2-chloro-benzoxazole and 2-chlorobenzothiozole, whereby the production of chlortetracycline is suppressed in favor of tetracycline.

4. A process as in claim 1 wherein from 0.2 to 100 parts per million of 2-ch1oro-benzoxazole is used.

5. A process as in claim 1 wherein from 0.2 to 100 parts per million of 2-chloro-benzothiozole is used.

6. In a process of aerobic fermentation of a nutrient medium with a microorganism of the genus Streptomyces, said fermentation normally producing an antibiotic consisting substantially of chlortet-racycline with minor amounts of tetracycline, the improvement comprising suppressing the production of chlortetracycline in favor of tetracycline by adding to the fermentation medium 0.2-100 parts per million, based on the weight of the broth, of a compound selected from the group consisting of Z-chIoro-benzoxazole and Z-chloro-benzothiazole.

References Cited in the file of this patent UNITED STATES PATENTS Gourevitch et al July 8, 1956 Minieri et al Feb. 7, 1956 FOREIGN PATENTS Great Britain Aug. 28, 1957 Switzerland Nov. 15, 1956 OTHER REFERENCES Martell et 81.: Chemistry of the Metal Chelate Compounds, 1952, Prentice Hall, Inc., Edgewood Cliffs, NJ., 60 pp. 139 to .149, 471.

Minieri et al.; Antibiotics Annual, 1953-1954. Sekizawa: I our. of Biochemistry, vol. 42, No. 2, March 1955, pp. 217-219. 

1. IN A PROCESS FOR PRODUCING TETRACYCLINE BY AEROBIC FERMENTATION WITH A CHLORTETRACYCLINE-PRODUCING MICROORGANISM OF THE GENUS STREPTOMYCES, THE IMPROVEMENT WHICH COMPRISES CONDUCTING THE FERMENTATION IN THE PRESENCE OF A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 2CHLORO-BENZOXAZOLE AND 2-CHLORO-BENZOTHIOZOLE, WHEREBY THE PRODUCTION OF CHLORTETRACYCLINE IS SUPPRESSED IN FAVOR OF TETRACYCLINE. 